Distance-Hi-C: Creating Photo Activated X-linkers To Define Nuclear Architecture

 描述(申请人提供)：DNA的核组织是复杂的，由多层组成。在最低分辨率下，大片染色体被集中在区域内，在较高水平上，染色体区域被组织在拓扑相关的区域中，这些区域提供了与启动子和远端调控元件(增强子)结合的转录因子(TF)相互作用的框架，最后调控因子、RNA聚合酶II和染色质的结构相互作用最终导致调控基因的表达。近年来，许多称为染色质(构象)捕获(CC)的方法被开发并用于捕获动态和稳定的DNA接触，这些接触构成了基因组结构和潜在的调控相互作用。这些方法的一个主要局限性是它们都依赖于单一的交联剂甲醛，它将DNA与蛋白质以及蛋白质与其他蛋白质进行交联。这使得对观察到的‘DNA-DNA’接触的解释变得复杂，而且缺乏距离信息。在这里，我们提出了一种名为距离-高-C(D-Hi-C)的正交化策略，在该策略中，我们设计、测试和应用了一组光激活的交联剂，旨在直接测量基因组范围内相互作用部位之间的距离。这些二价交联剂由两个由连接基隔开的活性基团组成。可以通过光激活进行交联的DNA插入物(即补骨脂素)将被用作反应基团。连接子将具有不同的精确长度，并且本质上是柔性的或刚性的，因此可以推断出交联基因座之间的三维距离。这种DNA特异的二价交联剂，当在Hi-C方案中取代甲醛时，产生空间限制，揭示增强子-启动子的相互作用，并潜在地以前所未有的精度推断核基因组的3D排列。此外，D-Hi-C有望降低背景，并允许检查被当前方法的高背景所掩盖的短期和中等范围的相互作用。除了在连接产物中加入生物素之外，在二价交联剂中添加诸如地高辛等基团将能够更好地纯化，从而更深入地检查基因组相互作用。最后，在基因激活后的时间内采样DNA距离提供了一种探索4D结构和在评估基因激活机制中设置关键限制的手段。其具体目标是1)合成一组二价交联剂，并评估它们在体外使DNA交联的能力；2)在果蝇核模型中测试D-Hi-C交联剂相对于甲醛的作用；3)将新的交联剂应用于Tier 1、ENCODE GM12878和K562细胞系，以测试它们在人类细胞中的有效性；以及4)测试特定于基因的光交联，这将使我们能够在基因激活的一段时间内更集中和更彻底地检查与基因组的基因位点特异性相互作用。这些方法的发展将广泛用于提供对基因调控机制的关键见解，这些机制在正常的动物发育和动态平衡中起作用，以及在癌症等疾病中出现问题。

 DESCRIPTION (provided by applicant): Nuclear organization of DNA is complex and consists of multiple layers. At the lowest resolution, large sections of chromosomes are packed in territories, at a higher level chromosomal regions are organized in topologically-associated domains that provide a framework for interactions of transcription factors (TFs) bound to promoters and distal regulatory elements (enhancers), and finally the structural interplay of regulators, RNA polymerase II, and chromatin then lead to regulated gene expression. In recent years, a number of methods called chromatin (conformation) capture (CC) have been developed and used to capture dynamic and stable DNA contacts that constitute genome architecture and potential regulatory interactions. A major limitation of these methods is that they all depend on a single crosslinker, formaldehyde, which crosslinks DNA to proteins as well as proteins to other proteins. This complicates the interpretation of the observed 'DNA-DNA' contacts and it lacks distance information. Here we propose an orthogonal strategy, named Distance-Hi-C (D-Hi-C), where we design, test, and apply a battery of photo-activated crosslinkers, designed to directly measure distances between interacting sites genome-wide. These bivalent crosslinkers consist of two reactive groups separated by a linker. DNA intercalaters that can be crosslinked by photo-activation (i.e. Psoralen) will be used as the reactive groups. Linkers will be of varying precise lengths, and either flexible or rigid in nature so that the 3-dimensional distance between crosslinked loci can be inferred. Such DNA-specific bivalent crosslinking reagents, when substituted for formaldehyde in Hi-C protocol, produces space constraints revealing enhancer- promoter interactions and potentially allowing the inference of the 3D arrangement of the nuclear genome with unprecedented precision. Moreover, D-Hi-C is expected to lower backgrounds and allow examination of short and moderate range interactions, which are obscured by high backgrounds of current methods. Addition of groups such as digoxigenin to the bivalent crosslinkers, in addition to biotin incorporated to the ligation products, will enable better purification and thus deeper examination of genomic interactions. Finally, sampling DNA distances in time following gene activation provides a means of exploring the 4D architecture and setting critical limits in evaluating mechanisms of gene activation. The specific aims are to 1) synthesize a battery of bivalent crosslinkers and evaluate their ability to crosslink DNA in vitro; 2) test D-Hi-C crosslinkers relative to formaldehyde in the Drosophila nuclei model; 3) apply new crosslinkers to tier 1, ENCODE GM12878 and K562 cell lines to test their efficacy in human cells; and 4) test locus-specific photo-crosslinking that will allow a more focused and thorough examination of locus-specific interactions with the genome in a time course of gene activation. The development of these methodologies will be broadly useful in providing critical insights into gene regulatory mechanisms that are operative in normal animal development and homeostasis and that go awry in diseases like cancer.